Maria Teresa Barbani

Interactions of Respiratory Viruses and the Nasal Microbiota during the First Year of Life in Healthy Infants

Respiratory viral infections are very frequent in infancy and of importance in acute and chronic disease development. Infections with human rhinovirus (HRV) are, e.g., associated with the later development of asthma. We found that only symptomatic HRV infections were associated with acute changes in the nasal microbiota, mainly characterized by a loss of microbial diversity. Infants with more frequent symptomatic HRV infections had a lower bacterial diversity at the end of the first year of life. Whether the interaction between viruses and the microbiota is one pathway contributing to asthma development will be assessed in the follow-ups of these children. Independent of that, measurements of microbial diversity might represent a potential marker for risk of later lung disease or monitoring of early life interventions. ABSTRACT Traditional culture techniques have shown that increased bacterial colonization is associated with viral colonization; however, the influence of viral colonization on the whole microbiota composition is less clear. We thus aimed to understand the interaction of viral infections and the nasal microbiota in early life to appraise their roles in disease development. Thirty-two healthy, unselected infants were included in this prospective longitudinal cohort study within the first year of life. Biweekly nasal swabs (n = 559) were taken, and the microbiota was analyzed by 16S rRNA pyrosequencing, and 10 different viruses and 2 atypical bacteria were characterized by real-time PCR (combination of seven duplex samples). In contrast to asymptomatic human rhinovirus (HRV) colonization, symptomatic HRV infections were associated with lower alpha diversity (Shannon diversity index [SDI]), higher bacterial density (PCR concentration), and a difference in beta diversities (Jaccard and Bray-Curtis index) of the microbiota. In addition, infants with more frequent HRV infections had a lower SDI at the end of the study period. Overall, changes in the microbiota associated with symptomatic HRV infections were characterized by a loss of microbial diversity. The interaction between HRV infections and the nasal microbiota in early life might be of importance for later disease development and indicate a potential approach for future interventions. IMPORTANCE Respiratory viral infections are very frequent in infancy and of importance in acute and chronic disease development. Infections with human rhinovirus (HRV) are, e.g., associated with the later development of asthma. We found that only symptomatic HRV infections were associated with acute changes in the nasal microbiota, mainly characterized by a loss of microbial diversity. Infants with more frequent symptomatic HRV infections had a lower bacterial diversity at the end of the first year of life. Whether the interaction between viruses and the microbiota is one pathway contributing to asthma development will be assessed in the follow-ups of these children. Independent of that, measurements of microbial diversity might represent a potential marker for risk of later lung disease or monitoring of early life interventions.

Sensitive and rapid detection of ganciclovir resistance by PCR based MALDI-TOF analysis

BACKGROUND Cytomegalovirus (CMV) infection is associated with significant morbidity and mortality in transplant recipients. Resistance against ganciclovir is increasingly observed. According to current guidelines, direct drug resistance testing is not always performed due to high costs and work effort, even when resistance is suspected. OBJECTIVES To develop a more sensitive, easy applicable and cost-effective assay as proof of concept for direct drug resistance testing in CMV surveillance of post-transplant patients. STUDY DESIGN Five consecutive plasma samples from a heart transplant patient with a primary CMV infection were analyzed by quantitative real-time polymerase chain reaction (rtPCR) as a surrogate marker for therapy failure, and by direct drug resistance detection assays such as Sanger sequencing and the novel primer extension (PEX) reaction matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) based method. RESULTS This report demonstrates that PEX reaction followed by MALDI-TOF analysis detects the A594V mutation, encoding ganciclovir resistance, ten days earlier compared to Sanger sequencing and more than 30 days prior to an increase in viral load. CONCLUSION The greatly increased sensitivity and rapid turnaround-time combined with easy handling and moderate costs indicate that this procedure could make a major contribution to improve transplantation outcomes.

Immunofluorescence versus xTAG multiplex PCR for the detection of respiratory picornavirus infections in children.

Abstract Background Polymerase chain reaction (PCR) is a sensitive tool for detection of respiratory picornaviruses. However, the clinical relevance of picornavirus detection by PCR is unclear. Immunofluorescence (IF), widely used to detect other respiratory viruses, has recently been introduced as a promising detection method for respiratory picornaviruses. Objectives To compare the clinical manifestations of respiratory picornavirus infections detected by IF with those of respiratory picornavirus infections detected by xTAG multiplex PCR in hospitalized children. Study design During a 1-year period, nasopharyngeal aspirates (NPA) from all children hospitalized due to an acute respiratory infection were prospectively analyzed by IF. All respiratory picornavirus positive IF samples and 100 IF negative samples were further tested with xTAG multiplex PCR. After exclusion of children with co-morbidities and viral co-infections, monoinfections with respiratory picornaviruses were detected in 108 NPA of 108 otherwise healthy children by IF and/or PCR. We compared group 1 children (IF and PCR positive, n =84) with group 2 children (IF negative and PCR positive, n =24) with regard to clinical manifestations of the infection. Results Wheezy bronchitis was diagnosed more often in group 1 than in group 2 (71% vs. 46%, p =0.028). In contrast, group 2 patients were diagnosed more frequently with pneumonia (17% vs. 6%, p =0.014) accompanied by higher levels of C-reactive protein (46mg/l vs. 11mg/l, p =0.009). Conclusions Picornavirus detection by IF in children with acute respiratory infection is associated with the clinical presentation of wheezy bronchitis. The finding of a more frequent diagnosis of pneumonia in picornavirus PCR positive but IF negative children warrants further investigation.

Rapid detection of respiratory picornaviruses in nasopharyngeal aspirates by immunofluorescence assay

Abstract Background Respiratory picornaviruses (enteroviruses and rhinoviruses) are commonly cited as causes of self-limited upper respiratory tract infection. However, it has recently been suggested that they may cause more severe respiratory disease. Immunofluorescence (IF) assays are rapid and inexpensive and are often used for the detection of respiratory viruses. Objectives We sought to develop an IF procedure, using commercially available reagents, for the detection of respiratory picornaviruses directly from nasopharyngeal aspirates (NPA). Study design From 1st November 2006 until 31st October 2007 all NPA from patients with respiratory infection were stained with the Light Diagnostic Pan-Enterovirus Reagent – “Blend” by IF (IF-ENVPAN). Those specimens which tested positive with this stain were further tested (subject to the availability of frozen specimen) with the xTAG respiratory viral panel, a multiplex PCR directed against respiratory picornaviruses, adenovirus (ADV), respiratory sincytial virus (RSV), influenza viruses A and B (IFA and IFB), parainfluenza virus (PIV) 1–4, human metapneumovirus (HMPV) and coronaviruses. Results 241/1122 NPA tested positive by IF-ENVPAN. 143 NPA were available for testing by xTAG respiratory viral panel. The multiplex PCR detected respiratory picornaviruses in 139 NPA, in 126 as the sole viral pathogen. Conclusions Our results indicate the potential of IF-ENVPAN for the laboratory detection of respiratory picornaviruses in clinical specimens. As far as we are aware, this is the first publication of such a method.

Respiratory viruses in healthy infants and infants with cystic fibrosis. A prospective cohort study

Rationale Acute viral respiratory tract infections in children with cystic fibrosis (CF) are known causes of disease exacerbation. The role of viral infections during infancy is, however, less known, although early infancy is thought to be a crucial period for CF disease development. We prospectively assessed symptomatic and asymptomatic viral detection in the first year of life in infants with CF and healthy controls. Methods In a prospective cohort study, we included 31 infants with CF from the Swiss Cystic Fibrosis Infant Lung Development Cohort and 32 unselected, healthy infants from the Basel Bern Infant Lung Development Cohort and followed them throughout the first year of life. Respiratory symptoms were assessed by weekly telephone interviews. Biweekly nasal swabs were analysed for 10 different viruses and two atypical bacteria with real-time seven duplex PCR (CF=561, controls=712). Measurements and results Infants with CF and healthy controls showed similar numbers of swabs positive for virus (mean 42% vs 44%; OR 0.91, 95% CI 0.66 to 1.26, p=0.6). Virus-positive swabs were less often accompanied by respiratory symptoms in infants with CF (17% vs 23%; OR 0.64, 95% CI 0.43 to 0.95, p=0.026). This finding was pronounced for symptomatic human rhinovirus detection (7% vs 11%; OR 0.52, 95% CI 0.31 to 0.9, p=0.02). Conclusions Viral detection is not more frequent in infants with CF and respiratory symptoms during viral detection occur even less often than in healthy controls. It is likely an interplay of different factors such as local epithelial properties and immunological mechanisms that contribute to our findings.

Lactate dehydrogenase concentration in nasal wash fluid indicates severity of rhinovirus-induced wheezy bronchitis in preschool children.

The clinical course of rhinovirus (RV)-associated wheezing illnesses is difficult to predict. We measured lactate dehydrogenase concentrations, RV load, antiviral and proinflammatory cytokines in nasal washes obtained from 126 preschool children with RV wheezy bronchitis. lactate dehydrogenase values were inversely associated with subsequent need for oxygen therapy. lactate dehydrogenase may be a useful biomarker predicting disease severity in RV wheezy bronchitis.